{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T00:36:47Z","timestamp":1792629407053,"version":"4.5.0"},"reference-count":63,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2022,1,20]],"date-time":"2022-01-20T00:00:00Z","timestamp":1642636800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>The routine techniques currently applied for the determination of nicotine and its major metabolites, cotinine, and trans-3\u2032-hydroxycotinine, in biological fluids, include spectrophotometric, immunoassays, and chromatographic techniques. The aim of this study was to develop, and compare two new chromatographic methods high-performance liquid chromatography coupled to triple quadrupole mass spectrometry (HPLC-QQQ-MS\/MS), and RP-HPLC enriched with chaotropic additives, which would allow reliable confirmation of tobacco smoke exposure in toxicological and epidemiological studies. The concentrations of analytes were determined in human plasma as the sample matrix. The methods were compared in terms of the linearity, accuracy, repeatability, detection and quantification limits (LOD and LOQ), and recovery. The obtained validation parameters met the ICH requirements for both proposed procedures. However, the limits of detection (LOD) were much better for HPLC-QQQ-MS\/MS (0.07 ng mL\u22121 for trans-3\u2032-hydroxcotinine; 0.02 ng mL\u22121 for cotinine; 0.04 ng mL\u22121 for nicotine) in comparison to the RP-HPLC-DAD enriched with chaotropic additives (1.47 ng mL\u22121 for trans-3\u2032-hydroxcotinine; 1.59 ng mL\u22121 for cotinine; 1.50 ng mL\u22121 for nicotine). The extraction efficiency (%) was concentration-dependent and ranged between 96.66% and 99.39% for RP-HPLC-DAD and 76.8% to 96.4% for HPLC-QQQ-MS\/MS. The usefulness of the elaborated analytical methods was checked on the example of the analysis of a blood sample taken from a tobacco smoker. The nicotine, cotinine, and trans-3\u2032-hydroxycotinine contents in the smoker\u2019s plasma quantified by the RP-HPLC-DAD method differed from the values measured by the HPLC-QQQ-MS\/MS. However, the relative errors of measurements were smaller than 10% (6.80%, 6.72%, 2.04% respectively).<\/jats:p>","DOI":"10.3390\/molecules27030682","type":"journal-article","created":{"date-parts":[[2022,1,20]],"date-time":"2022-01-20T22:51:06Z","timestamp":1642719066000},"page":"682","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":18,"title":["Application of HPLC-QQQ-MS\/MS and New RP-HPLC-DAD System Utilizing the Chaotropic Effect for Determination of Nicotine and Its Major Metabolites Cotinine, and trans-3\u2032-Hydroxycotinine in Human Plasma Samples"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1372-8987","authenticated-orcid":false,"given":"Jacek","family":"Baj","sequence":"first","affiliation":[{"name":"Department of Anatomy, Medical University of Lublin, Jaczewskiego 4, 20-090 Lublin, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0509-868X","authenticated-orcid":false,"given":"Wojciech","family":"Flieger","sequence":"additional","affiliation":[{"name":"Department of Anatomy, Medical University of Lublin, Jaczewskiego 4, 20-090 Lublin, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dominika","family":"Przygodzka","sequence":"additional","affiliation":[{"name":"Department of Forensic Medicine, Medical University of Lublin, 20-090 Lublin, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7637-6387","authenticated-orcid":false,"given":"Grzegorz","family":"Buszewicz","sequence":"additional","affiliation":[{"name":"Department of Forensic Medicine, Medical University of Lublin, 20-090 Lublin, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4184-9305","authenticated-orcid":false,"given":"Grzegorz","family":"Teresi\u0144ski","sequence":"additional","affiliation":[{"name":"Department of Forensic Medicine, Medical University of Lublin, 20-090 Lublin, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6660-1384","authenticated-orcid":false,"given":"Magdalena","family":"Pizo\u0144","sequence":"additional","affiliation":[{"name":"Department of Analytical Chemistry, Medical University of Lublin, Chod\u017aki 4A, 20-093 Lublin, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2115-9649","authenticated-orcid":false,"given":"Ryszard","family":"Maciejewski","sequence":"additional","affiliation":[{"name":"Department of Anatomy, Medical University of Lublin, Jaczewskiego 4, 20-090 Lublin, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6881-3161","authenticated-orcid":false,"given":"Jolanta","family":"Flieger","sequence":"additional","affiliation":[{"name":"Department of Analytical Chemistry, Medical University of Lublin, Chod\u017aki 4A, 20-093 Lublin, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,1,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1055\/s-0029-1224138","article-title":"Neuroprotective and neurotoxic effects of nicotine","volume":"42","author":"Ferrea","year":"2009","journal-title":"Pharmacopsychiatry"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1007\/s00204-013-1127-0","article-title":"How much nicotine kills a human? Tracing back the generally accepted lethal dose to dubious self-experiments in the nineteenth century","volume":"88","author":"Mayer","year":"2014","journal-title":"Arch. Toxicol."},{"key":"ref_3","unstructured":"(2019, January 31). Available online: https:\/\/www.rch.org.au\/clinicalguide\/guideline_index\/Nicotine_Poisoning\/."},{"key":"ref_4","unstructured":"Rodgman, A., and Perfetti, T.A. (2013). The Chemical Components of Tobacco and Tobacco Smoke, CRC Press, Taylor & Francis Group. [2nd ed.]."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1672","DOI":"10.1093\/jnci\/djn389","article-title":"Annual report to the nation on the status of cancer, 1975\u20132005, featuring trends in lung cancer, tobacco use, and tobacco control","volume":"100","author":"Jemal","year":"2008","journal-title":"J. Natl. Cancer Inst."},{"key":"ref_6","unstructured":"(2009). WHO Report on the Global Tobacco Epidemic 2009: Implementing Smoke-Free Environments, World Health Organization."},{"key":"ref_7","unstructured":"(2021). WHO Report on the Global Tobacco Epidemic 2021: Addressing New and Emerging Products, World Health Organization."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1146\/annurev.pharmtox.48.113006.094742","article-title":"Pharmacology of nicotine: Addiction, smoking-induced disease, and therapeutics","volume":"49","author":"Benowitz","year":"2009","journal-title":"Annu. Rev. Pharmacol. Toxicol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1007\/978-3-540-69248-5_2","article-title":"Nicotine chemistry, metabolism, kinetics and biomarkers","volume":"192","author":"Benowitz","year":"2009","journal-title":"Handb. Exp. Pharmacol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1124\/pr.57.1.3","article-title":"Metabolism and disposition kinetics of nicotine","volume":"57","author":"Hukkanen","year":"2005","journal-title":"Pharmacol. Rev."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"619","DOI":"10.1016\/j.toxicon.2004.01.017","article-title":"Nicotine, its metabolism and an overview of its biological effects","volume":"43","author":"Yildiz","year":"2004","journal-title":"Toxicon"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1007\/978-3-319-13665-3_3","article-title":"Pharmacogenetics of nicotine and associated smoking behaviors","volume":"23","author":"Tanner","year":"2015","journal-title":"Curr. Top. Behav. Neurosci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1093\/oxfordjournals.epirev.a017925","article-title":"Cotinine as a biomarker of environmental tobacco smoke exposure","volume":"18","author":"Benowitz","year":"1996","journal-title":"Epidemiol. Rev."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"487","DOI":"10.1023\/A:1024672522802","article-title":"Serum cotinine as a marker of environmental tobacco smoke exposure in epidemiological studies: The experience of the MATISS project","volume":"18","author":"Seccareccia","year":"2003","journal-title":"Eur. J. Epidemiol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"639","DOI":"10.1021\/tx990015e","article-title":"Characterization of multiple products of cytochrome P450 2A6-catalyzed cotinine metabolism","volume":"12","author":"Murphy","year":"1999","journal-title":"Chem. Res. Toxicol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Flieger, J., Kawka, J., and Tatarczak-Michalewska, M. (2019). Levels of the Thiocyanate in the Saliva of Tobacco Smokers in Comparison to e-Cigarette Smokers and Nonsmokers Measured by HPLC on a Phosphatidylcholine Column. Molecules, 24.","DOI":"10.3390\/molecules24203790"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1598","DOI":"10.1093\/clinchem\/31.10.1598","article-title":"Analysis for nicotine and cotinine in hair to determine cigarette smoker status","volume":"31","author":"Haley","year":"1985","journal-title":"Clin. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"979","DOI":"10.1093\/clinchem\/32.6.979","article-title":"Quantification of cotinine in plasma and saliva by liquid chromatography","volume":"32","author":"Machacek","year":"1986","journal-title":"Clin. Chem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"699","DOI":"10.2105\/AJPH.78.6.699","article-title":"Cotinine in the serum, saliva, and urine of nonsmokers, passive smokers, and active smokers","volume":"78","author":"Wall","year":"1988","journal-title":"Am. J. Public Health"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1238","DOI":"10.1016\/j.cca.2010.04.027","article-title":"A sensitive enzyme immunoassay for measuring cotinine in passive smokers","volume":"411","author":"Park","year":"2010","journal-title":"Clin. Chim. Acta"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1016\/0378-4347(93)80103-B","article-title":"Determination of nicotine and four metabolites in the serum of smokers by high-performance liquid chromatography with ultraviolet detection","volume":"621","author":"Zuccaro","year":"1993","journal-title":"J. Chromatogr."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/j.jchromb.2013.06.028","article-title":"Simultaneous determination of nicotine and cotinine in serum using high-performance liquid chromatography with fluorometric detection and postcolumn UV-photoirradiation system","volume":"934","author":"Yasuda","year":"2013","journal-title":"J. Chromatogr. B Analyt. Technol. Biomed. Life Sci."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Avagyan, R., Spasova, M., and Lindholm, J. (2021). Determination of Nicotine-Related Impurities in Nicotine Pouches and Tobacco-Containing Products by Liquid Chromatography\u2013Tandem Mass Spectrometry. Separations, 8.","DOI":"10.3390\/separations8060077"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"6473","DOI":"10.1007\/s00216-016-9766-7","article-title":"Simultaneous determination of nicotine, cotinine, and nicotine N-oxide in human plasma, semen, and sperm by LC-Orbitrap MS","volume":"408","author":"Arafat","year":"2016","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1016\/j.jchromb.2009.11.032","article-title":"Determination of nicotine and cotinine in human serum by means of LC\/MS","volume":"878","author":"Baumann","year":"2010","journal-title":"J. Chromatogr. B Analyt. Technol. Biomed. Life Sci."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1016\/j.jchromb.2010.12.012","article-title":"Determination of the nicotine metabolites cotinine and trans-3\u2032-hydroxycotinine in biologic fluids of smokers and non-smokers using liquid chromatography-tandem mass spectrometry: Biomarkers for tobacco smoke exposure and for phenotyping cytochrome P450 2A6 activity","volume":"879","author":"Jacob","year":"2011","journal-title":"J. Chromatogr. B Analyt. Technol. Biomed. Life Sci."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1124","DOI":"10.1002\/bmc.1581","article-title":"A liquid chromatography-mass spectrometry method for nicotine and cotinine; utility in screening tobacco exposure in patients taking amiodarone","volume":"25","author":"Gabr","year":"2011","journal-title":"Biomed. Chromatogr."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2349","DOI":"10.1007\/s00216-009-3157-2","article-title":"Optimization and validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of nicotine, cotinine, trans-3\u2032-hydroxycotinine and norcotinine in human oral fluid","volume":"395","author":"Shakleya","year":"2009","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"3574","DOI":"10.1016\/j.jchromb.2011.09.046","article-title":"Quantification of nicotine, cotinine, trans-3\u2032-hydroxycotinine and varenicline in human plasma by a sensitive and specific UPLC-tandem mass-spectrometry procedure for a clinical study on smoking cessation","volume":"879","author":"Dobrinas","year":"2011","journal-title":"J. Chromatogr. B Analyt. Technol. Biomed. Life Sci."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"978","DOI":"10.1016\/j.cca.2012.02.017","article-title":"Simultaneous quantification of nicotine, cotinine, trans-3\u2032-hydroxycotinine, norcotinine and mecamylamine in human urine by liquid chromatography-tandem mass spectrometry","volume":"413","author":"Scheidweiler","year":"2012","journal-title":"Clin. Chim. Acta"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"485","DOI":"10.1016\/j.pupt.2007.11.001","article-title":"GC-MS determined cotinine in an epidemiological study on smoking status at delivery","volume":"21","author":"Chazeron","year":"2008","journal-title":"Pulm. Pharmacol. Ther."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"322","DOI":"10.1016\/j.jchromb.2006.07.029","article-title":"Simple, rapid and sensitive assay method for simultaneous quantification of urinary nicotine and cotinine using gas chromatography-mass spectrometry","volume":"844","author":"Man","year":"2006","journal-title":"J. Chromatogr. B Analyt. Technol. Biomed. Life Sci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"389","DOI":"10.1007\/978-1-60761-459-3_38","article-title":"Quantitation of nicotine, its metabolites, and other related alkaloids in urine, serum, and plasma using LC-MS-MS","volume":"603","author":"Yue","year":"2010","journal-title":"Methods Mol. Biol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2323","DOI":"10.1373\/clinchem.2004.038489","article-title":"Simultaneous and sensitive measurement of anabasine, nicotine, and nicotine metabolites in human urine by liquid chromatography-tandem mass spectrometry","volume":"50","author":"Xu","year":"2004","journal-title":"Clin. Chem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"390","DOI":"10.1021\/acs.chemrestox.5b00521","article-title":"Quantitation of the Minor Tobacco Alkaloids Nornicotine, Anatabine, and Anabasine in Smokers\u2019 Urine by High Throughput Liquid Chromatography-Mass Spectrometry","volume":"29","author":"Thomson","year":"2016","journal-title":"Chem. Res. Toxicol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"882","DOI":"10.1158\/1055-9965.EPI-18-0018","article-title":"A Comparison of Direct and Indirect Analytical Approaches to Measuring Total Nicotine Equivalents in Urine","volume":"27","author":"Taghavi","year":"2018","journal-title":"Cancer Epidemiol. Biomark. Prev."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"3617","DOI":"10.31557\/APJCP.2019.20.12.3617","article-title":"Quantification of Nicotine and Cotinine in Plasma, Saliva, and Urine by HPLC Method in Chewing Tobacco Users","volume":"20","author":"Shaik","year":"2019","journal-title":"Asian Pac. J. Cancer Prev."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"725","DOI":"10.1016\/j.jchromb.2009.12.018","article-title":"A novel validated procedure for the determination of nicotine, eight nicotine metabolites and two minor tobacco alkaloids in human plasma or urine by solid-phase extraction coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry","volume":"878","author":"Miller","year":"2010","journal-title":"J. Chromatogr. B"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1016\/S0378-4347(01)00451-0","article-title":"Simultaneous determination of nicotine and eight nicotine metabolites in urine of smokers using liquid chromatography-tandem mass spectrometry","volume":"778","author":"Meger","year":"2002","journal-title":"J. Chromatogr. B"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"McGuffey, J.E., Wei, B., Bernert, J.T., Morrow, J.C., Xia, B., Wang, L., and Blount, B.C. (2014). Validation of a LC-MS\/MS method for quantifying urinary nicotine, six nicotine metabolites and the minor tobacco alkaloids\u2014Anatabine and anabasine\u2014In smokers\u2019 urine. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0101816"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"7528","DOI":"10.1016\/j.chroma.2010.10.005","article-title":"Determination of nicotine and nicotine metabolites in urine by hydrophilic interaction chromatography-tandem mass spectrometry: Potential use of smokeless tobacco products by ice hockey players","volume":"1217","author":"Marclay","year":"2010","journal-title":"J. Chromatogr. A"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"108","DOI":"10.1016\/j.jpba.2008.09.044","article-title":"Determination of nicotine, cotinine, and related alkaloids in human urine and saliva by automated in-tube solid-phase microextraction coupled with liquid chromatography-mass spectrometry","volume":"49","author":"Kataoka","year":"2009","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"312","DOI":"10.1002\/bmc.463","article-title":"Validation and application of a method for the determination of nicotine and five major metabolites in smokers\u2019 urine by solid-phase extraction and liquid chromatography-tandem mass spectrometry","volume":"19","author":"Heavner","year":"2005","journal-title":"Biomed. Chromatogr."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"290","DOI":"10.1016\/j.cca.2014.06.012","article-title":"A high-throughput robotic sample preparation system and HPLC-MS\/MS for measuring urinary anatabine, anabasine, nicotine and major nicotine metabolites","volume":"436","author":"Wei","year":"2014","journal-title":"Clin. Chim. Acta"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"2394","DOI":"10.1002\/jssc.201300220","article-title":"A simple, fast, and sensitive method for the measurement of serum nicotine, cotinine, and nornicotine by LC-MS\/MS","volume":"36","author":"Yuan","year":"2013","journal-title":"J. Sep. Sci."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1016\/j.jchromb.2013.03.032","article-title":"Simultaneous serum nicotine, cotinine, and trans-3\u2032-hydroxycotinine quantitation with minimal sample volume for tobacco exposure status of solid organ transplant patients","volume":"928","author":"Shu","year":"2013","journal-title":"J. Chromatogr. B"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1093\/chromsci\/43.3.133","article-title":"A rapid LC-MS-MS method for the determination of nicotine and cotinine in serum and saliva samples from smokers: Validation and comparison with a radioimmunoassay method","volume":"43","author":"Byrd","year":"2005","journal-title":"J. Chromatogr. Sci."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1016\/j.jchromb.2016.03.020","article-title":"A fully validated LC-MS\/MS method for simultaneous determination of nicotine and its metabolite cotinine in human serum and its application to a pharmacokinetic study after using nicotine transdermal delivery systems with standard heat application in adult smokers","volume":"1020","author":"Abdallah","year":"2016","journal-title":"J. Chromatogr. B"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"258","DOI":"10.1177\/0960327111422401","article-title":"Hair nicotine\/cotinine concentrations as a method of monitoring exposure to tobacco smoke among infants and adults","volume":"31","author":"Tzatzarakis","year":"2012","journal-title":"Hum. Exp. Toxicol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"321","DOI":"10.1093\/anatox\/35.6.321","article-title":"Validation of a liquid chromatography-tandem mass spectrometry method for the detection of nicotine biomarkers in hair and an evaluation of wash procedures for removal of environmental nicotine","volume":"35","author":"Miller","year":"2011","journal-title":"J. Anal. Toxicol."},{"key":"ref_51","doi-asserted-by":"crossref","unstructured":"Kim, J., Cho, H.D., Suh, J.H., Lee, J.Y., Lee, E., Jin, C.H., Wang, Y., Cha, S., Im, H., and Han, S.B. (2020). Analysis of Nicotine Metabolites in Hair and Nails Using QuEChERS Method Followed by Liquid Chromatography-Tandem Mass Spectrometry. Molecules, 25.","DOI":"10.3390\/molecules25081763"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"272","DOI":"10.1016\/j.jpba.2018.04.032","article-title":"Analysis of nicotine and cotinine in hair by on-line in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry as biomarkers of exposure to tobacco smoke","volume":"156","author":"Inukai","year":"2018","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"655","DOI":"10.1002\/bmc.369","article-title":"Hair analysis of nicotine and cotinine for evaluating tobacco smoke exposure by liquid chromatography-mass spectrometry","volume":"18","author":"Chetiyanukornkul","year":"2004","journal-title":"Biomed. Chromatogr."},{"key":"ref_54","unstructured":"Xia, B., McGuffey, J., Xia, Y., Guillot, T., McGahee, E., Wang, L., and Blount, B. (2017). Sensitive, Rapid and High Throughput Measurement of Nicotine in Human Serum by Automation and Liquid Chromatography-Atmospheric Pressure Ionization Tandem Mass Spectrometry, American Chemical Society."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"13962","DOI":"10.1021\/acsomega.0c05696","article-title":"Sensitive Quantification of Nicotine in Bronchoalveolar Lavage Fluid by Acetone Precipitation Combined with Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry","volume":"6","author":"Xia","year":"2021","journal-title":"ACS Omega"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1016\/j.chroma.2006.01.090","article-title":"The effect of chaotropic mobile phase additives on the separation of selected alkaloids in reversed-phase high-performance liquid chromatography","volume":"1113","author":"Flieger","year":"2006","journal-title":"J. Chromatogr. A"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"733","DOI":"10.1002\/jssc.201000797","article-title":"Comparison of chaotropic salt and ionic liquid as mobile phase additives in reversed-phase high-performance liquid chromatography of biogenic amines","volume":"34","author":"Flieger","year":"2011","journal-title":"J. Sep. Sci."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"469","DOI":"10.1002\/jssc.201200798","article-title":"Usefulness of chaotropic salt additive in RP-HPLC of organic nonionized compounds","volume":"36","author":"Flieger","year":"2013","journal-title":"J. Sep. Sci."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1039\/C2AY25678A","article-title":"Spectroscopic investigations into the acid\u2013base properties of nicotine at different temperatures","volume":"5","author":"Clayton","year":"2013","journal-title":"Anal. Methods"},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/bs.acc.2014.09.001","article-title":"Biomarkers of Tobacco Smoke Exposure","volume":"67","author":"Mattes","year":"2014","journal-title":"Adv. Clin. Chem."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"E109","DOI":"10.1208\/aapsj0901011","article-title":"Key elements of bioanalytical method validation for small molecules","volume":"9","author":"Bansal","year":"2007","journal-title":"AAPS J."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"1962","DOI":"10.1007\/s11095-007-9291-7","article-title":"Quantitative bioanalytical methods validation and implementation: Best practices for chromatographic and ligand binding assays","volume":"24","author":"Viswanathan","year":"2007","journal-title":"Pharm. Res."},{"key":"ref_63","doi-asserted-by":"crossref","unstructured":"Kim, S. (2016). Overview of Cotinine Cutoff Values for Smoking Status Classification. Int. J. Environ. Res. Public Health, 13.","DOI":"10.3390\/ijerph13121236"}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/27\/3\/682\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T22:04:59Z","timestamp":1760133899000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/27\/3\/682"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,1,20]]},"references-count":63,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2022,2]]}},"alternative-id":["molecules27030682"],"URL":"https:\/\/doi.org\/10.3390\/molecules27030682","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,1,20]]}}}